The invention described herein was made in the course of work supported in part by NCI Grant CA 21399 and in part by NIH Grant AM 28469.
It is well-known that some diseases have an autoimmune pathogenetic mechanism. Among these are rheumatoid arthritis, systemic lupus erythematosus, and chronic active hepatitis. A general increase in the level of gammaglobulins (antibodies) often accompanies an increase in the level of a particular antibody or antibodies in patient serum. These observations have led to the suggestion that polyclonal (general) activation of B-lymphocytes is involved in the disease process and might even precede the emergence of a particular antibody. For example, in rheumatoid arthritis the symptoms may appear before the characteristic antibody, i.e., the rheumatoid factor, develops.
On the other hand, other arthritides are known to have no systemic autoimmune basis and are commonly described as "sero-negative". These include Reiter's syndrome, ankylosing spondylitis, psoriatic arthritis, osteo-arthritis, and gout.
In each autoimmune disease the presence of one or several antibody specificities has been observed, but the presence of an antibody in the absence of disease has also been reported, particularly in elderly patients. Under these circumstances the role of antibodies in some autoimmune diseases has always been questionable, although they are currently used as disease indicators.
Another feature of autoimmune diseases is the presence of immune complexes, which, under certain conditions can activate the complement cascade. For example, it is usual for serum complement to be activated in systemic lupus erythematosus but very unusual in rheumatoid arthritis, although immune complexes have been described in both diseases. The immune complexes are expected to fix complement, resulting in the activation of proteolytic enzymes.
Aside from other proteases, two major systems generate proteolytic enzymes: the complement system mentioned above and the coagulation system. Most if not all of the enzymes of these two systems are known to require calcium ion (Ca.sup.++) for their function.
Another property of the proteolytic enzymes found in the serum is their ability to bind to .alpha..sub.1 antitrypsin, which inactivates the enzyme, or to .alpha..sub.2 -macroglobulin (.alpha..sub.2 M). The .alpha..sub.2 M is capable of binding the proteases but the active site remains free to act. The only restriction is that while the free enzyme can degrade both high and low molecular weight substrates, the enzyme bound to .alpha..sub.2 M can only degrade low molecular weight substrates. At the same time the free enzymes are blocked by large-molecule inhibitors such as soybean trypsin inhibitor (SBTI, m.w. 21,000) as well as by low-molecule weight inhibitors such as aprotinin, phenyl-methyl-sulfonyl-fluoride, or diisopropyl phosphofluoridate. It appears that SBTI has some inhibitory effect on .alpha..sub.2 M-bound trypsin but it is much less effective than the low m.w. inhibitors.
It has been reported in Int. J. Immunopharmac., vol. 4, no. 1, pp. 1-7 (1982), that an .alpha..sub.2 M-associated factor is produced in cultures of rabbit lymphoid cells. This factor (or lymphokine) has the ability to activate B lymphocytes, i.e., it was a polyclonal B-cell activator (PBA). Its activating effect was blocked by low m.w. protease inhibitors but not by SBTI. This PBA behaved as a trypsin-.alpha..sub.2 M complex. In the next step .alpha..sub.2 M was extracted from patient serum and from normal human serum and employed in determining directly the activity for tosyl-arginyl-methylester (TAME). The absorption at 247 nm after 30 min. at 37.degree. C. was determined. Slightly more proteolytic activity was observed with purified .alpha..sub.2 M from patient serum.
There remains a need for the determination of the proteolytic activity associated with .alpha..sub.2 M directly in human blood. Many problems are certain to be encountered in such a program. For example, there are many proteases in the serum, particularly from the coagulation system, that can bind to .alpha..sub.2 M; thus serum cannot be used. If Ca.sup.2+ blocking agents are used to obtain plasma, the enzyme may not be functional. If SBTI is used to block free proteases in plasma, it may slow and alter significantly the enzyme activity associated with .alpha..sub.2 M. If .alpha..sub.2 M is first attached to a solid phase or is precipitated by an antibody, it may carry with it proteases from complement or the coagulation system. No technique is readily suggested by the prior art and, indeed, it would appear from that art that the measurement of any significant amount of enzyme activity would not be possible because of difficulties in obtaining plasma suitable for the purpose.